Tackling Treg mediated resistance to radiation and anti-PDL1 in HNSCCs

摘要 当前使用免疫检查点抑制剂和免疫抑制剂治疗头颈部鳞状细胞癌（HNSCC） 放疗（RT）最初有效，但复发是常见的。我们研究的目的是了解 反应和抵抗机制，并利用这些知识来改善治疗。我们发现，在 在治疗反应阶段，RT增加STAT 1磷酸化、CXCL 9/10分泌和PDL 1 在癌细胞上表达，并增加CD 8和CD 4浸润和活化。然而，这些影响， 在抵抗期消失，调节性T细胞（TcR）、STAT 3和TGF β 1显著增加， 被观察到。Treg耗竭和抗STAT 3靶向恢复对RT和抗PDL 1的应答，导致 抑制TGF β 1的血浆水平，活化T效应细胞（Teff，Cd 44+，IFN-G+ CD 4和CD 8 T 细胞）和完全肿瘤根除。这种效果是无法实现的， 我们的体外初步数据表明，RT 增强CD 4 T细胞上的STAT 3磷酸化，导致Treg转化增加。这是颠倒与 STAT 3抑制剂。CD 4 T细胞向Treg表型的转化代表了CD 4 T细胞增殖的重要机制。 免疫逃避TGF β 1已被确定为幼稚CD 4 T细胞转化的必要生长因子。 细胞通过诱导FoxP 3表达而转化为TcB。此外，STAT 3是一种必需的转录辅因子， FoxP 3的表达。我们的中心假设是，在反应阶段，RT诱导了一个明显的 癌细胞STAT 1介导的CXCL 9/10分泌，其募集并激活Teff， 增强与抗PDL 1的协同作用。然而，RT也通过以下方式促进CD 4转化为Treg细胞： STAT 3的磷酸化，其通过抑制Teff而导致治疗抗性的发展 功能在癌细胞和CXCR 3-/-小鼠上使用STAT 1和CXCL 9/10的敲低构建体 模型，我们将确定RT诱导的趋化因子在Teff趋化性，增殖和TCR中的作用 激活（Aim 1）。为了确定RT如何影响CD 4 T细胞转化以及如何反馈到负面影响， 影响Teff功能，我们将使用CD 4-Cre/Stat 3flox/flox小鼠，同时敲低CD 4 T细胞上的STAT 3 STAT 3抑制剂（Aim 2）。最后，我们将使用来自I期临床试验的组织和血液样本， 使用新辅助组合RT和抗PDL 1，并将检查是否会改变免疫功能， 景观以增加Teff/Treg比率并增强Teff活化概况（Aim 3）。我们希望这些研究能够 阐明RT加免疫检查点阻断的分子和细胞参数，并帮助开发更多 有效治疗HNSCC。

Abstract Current treatment of head and neck squamous cell carcinoma (HNSCC) with immune checkpoint inhibitors and radiotherapy (RT) are initially effective, but relapse is common. The goal of our research is to understand mechanisms of response and resistance and to use this knowledge to improve therapy. We show that during the response phase to treatment, RT increases STAT1 phosphorylation, CXCL9/10 secretion, and PDL1 expression on cancer cells, and increases CD8 and CD4 infiltration and activation. These effects, however, dissipate in the resistance phase, and a significant increase in regulatory T cells (Tregs), STAT3, and TGF1 are observed. Treg depletion and anti-STAT3 targeting restores response to RT and anti-PDL1, resulting in suppression of plasma levels of TGF1, activation of T effector cells (Teff, Cd44+, IFN-G+ CD4 and CD8 T cells) and complete tumor eradication in an orthotopic model of HNSCC. This effect is unachievable with the addition of anti-CTLA4, anti-Tim3, or with hypofractionation of RT. Our in vitro preliminary data show that RT enhances STAT3 phosphorylation on CD4 T cells, leading to increased Treg conversion. This is reversed with STAT3 inhibitors. CD4 T cell conversion toward a Treg phenotype represents an important mechanism of immune evasion. TGF1 has been established as a necessary growth factor for conversion of naïve CD4 T cells to Tregs through induction of FoxP3 expression. In addition, STAT3 is a necessary transcription co-factor for FoxP3 expression. Our central hypothesis is that during the response phase, RT induces a distinct cancer cell STAT1-mediated CXCL9/10 secretion which recruits and activates Teff resulting in enhanced synergy with anti-PDL1. RT, however, also promotes CD4 conversion to Treg cells by phosphorylation of STAT3, which leads to the development of treatment resistance by inhibiting Teff function. Using knockdown constructs of STAT1 and CXCL9/10 on cancer cells and a CXCR3-/- mouse model, we will determine the effect of RT-induced chemokines in Teff chemotaxis, proliferation, and TCR activation (Aim1). To determine how RT affects CD4 T cell conversion and how that feeds back to negatively affect Teff function, we will use the CD4-Cre/ Stat3flox/flox mice with knockdown of STAT3 on CD4 T cells as well as STAT3 inhibitors (Aim2). Finally, we will use tissue and blood samples from a Phase I clinical trial aimed at using neoadjuvant combination RT and anti-PDL1 and will examine whether that changes the immune landscape to increase Teff/Treg ratio and enhance Teff activation profile (Aim3). We expect these studies to elucidate molecular and cellular parameters of RT plus immune checkpoint blockade and to help develop more effective therapy for HNSCC.